KR102326127B1 - Development of injectable tissue adhesive hydrogel based on polyphenol and its application - Google Patents

Abstract

The present invention relates to an injection-type hydrogel capable of providing a crosslinking point and physiological activity of a hydrogel with epigallocatechin gallate (EGCG), which is a green tea extract and capable of removing radicals in the body, a method for preparing the same, and a method for preparing the same It is about application.
In the present invention, an EGCG duplex was introduced into the hydrogel to impart rapid crosslinking and immune response modulating ability to the hydrogel. In addition, EGCG, known as a competitive inhibitor of tyrosinase, has significantly higher reactivity with tyrosinase than with tyrosine and tyrosinase, so Streptomyces avermitilis derived tyrosinase It was used as a crosslinking agent. The present invention has developed a hydrogel system capable of introducing an EGCG molecule into a hydrogel, which can be used as a cross-linking point, can impart tissue adhesion, and can control an immune response in the body.

Description

Injection-type tissue adhesive hydrogel using green tea-derived polyphenol, its preparation method, and its application {Development of injectable tissue adhesive hydrogel based on polyphenol and its application}

The present invention provides an injection-type hydrogel capable of providing physiological activity by using green tea extract and epigallocatechin gallate (EGCG), which can remove radicals in the body, as a crosslinking point of the hydrogel, and a method for preparing the same and to their applications.

The traditional purpose of hydrogels applied to tissue engineering and regenerative medicine has been to deliver growth factors or drugs into the body to provide physiological activity to surrounding tissues. Looking at the latest trends, multi-purpose hydrogels are being actively developed to solve some problems for applying biomaterials to actual clinical practice. Such versatility includes 1) it must have the ability to adhere to surrounding tissues, 2) it must achieve rapid cross-linking for minimally invasive delivery, 3) it must have self-healing ability to adapt to dynamic changes in the body, and finally 4) There is a need to be able to control the immune response to increase the success rate of transplantation in the body.

The actual skin tissue is one of the most mobile tissues in the human body, and after it is incised, it is necessary to prevent the incision from opening through strong adhesion. In addition, the skin tissue is the largest barrier that can separate the body from the immune system and the external environment. And after the skin tissue is adhered, regeneration should be induced into a tissue similar to the original tissue through tissue remodeling.

Currently, adhesives are used to bond hydrogels and implants to the body, but they have problems such as toxicity in the body, induction of immune responses, and low adhesion in wet environments. Specifically, fibrin glue and cyanoacrylate glue are most commonly used in the current medical field. However, fibrin glue induces an immune response and has several disadvantages, such as weak adhesion. In addition, cyanoacrylate glue has a strong adhesive ability, but has a disadvantage in that the adhesive ability is very poor in a wet environment.

Therefore, it is the most ideal method to impart adhesion to the material to be transplanted.

In addition, when the hydrogel enters the body, it is necessary to minimize the immune response and the foreign body reaction to increase the transplant success rate. In general, when a hydrogel is injected into the body to initiate an immune response, a foreign body reaction occurs and a fibrous membrane is formed around the implant to interfere with interaction with surrounding tissues. These reactions lead to graft failure and damage to surrounding tissues.

In addition, in order to apply the adhesive to the wound site of the tissue, it is important to induce an immune response to a minimum and maximize the success rate of the applied adhesive. It is known that EGCG induces a minimal immune response by removing radicals.

Recently, as the ability of green tea-derived EGCG to regulate immune response through radical removal in the body has been known, many attempts have been made to introduce EGCG into the body. Therefore, there is a need for a method for introducing and delivering EGCG into a biopolymer in order to enhance the effect of EGCG on a desired site.

Laid-Open Patent Publication No. 10-2016-0051839 (2016.05.11) Laid-open Patent Publication No. 10-2018-0046517 (2018.05.09) Laid-Open Patent Publication No. 10-2012-0049419 (2012.05.17)

An object of the present invention is to provide an adhesive hydrogel composition having a strong tissue adhesion ability by using the strong oxidative power of EGCG, and the ability to control an immune response by removing radicals in the body.

Another object of the present invention is to provide a method for preparing the adhesive hydrogel composition.

Another object of the present invention is to provide a hydrogel system for regulating tissue adhesion and immune response that can be delivered in an injectable form.

In order to solve the above problems, the present inventors introduced the EGCG duplex into the polymer in order to give the hydrogel fast crosslinking and immune response modulating ability.

Since EGCG, known as a competitive inhibitor of tyrosinase, has significantly higher reactivity with tyrosinase than with tyrosine and tyrosinase, tyrosinase derived from Streptomyces avermitilis was used as a crosslinking agent. .

In addition, the reaction by tyrosinase between the polymer and the EGCG polymer does not smoothly form a covalent bond due to the steric hindrance of EGCG. On the other hand, when a polymer-phenol polymer or polymer-phenol derivative polymer having a relatively small size is combined in a certain ratio, this steric hindrance can be overcome and a rapid crosslinking reaction can be induced.

Specifically, the present invention provides the following.

(1) a polymer obtained by introducing EGCG (epigallocatechin gallate) into hyaluronic acid, alginate, chondroitin sulfate, chitosan or polyethylene glycol; and

A polymer introduced with phenol or a phenol derivative is blended with hyaluronic acid, alginate, chondroitin sulfate, chitosan or polyethylene glycol,

Streptomyces avermitilis ( Streptomyces avermitilis ) A method for producing an adhesive hydrogel composition comprising the step of cross-linking using a tyrosinase derived.

(2) The method for producing an adhesive hydrogel composition according to (1), wherein a polymer obtained by introducing EGCG into hyaluronic acid and a polymer obtained by introducing a phenol derivative into hyaluronic acid are blended.

(3) The method for producing an adhesive hydrogel composition according to (1), wherein the phenol derivative is tyramine, tyrosine, 4-hydroxyphenylacetic acid or 3-(4-hydroxyphenyl)propionic acid.

(4) The method for producing an adhesive hydrogel composition according to (1), wherein the phenol derivative is tyramine or tyrosine.

(5) (1) to (4) an adhesive hydrogel composition prepared by any one of the manufacturing method.

(6) The adhesive hydrogel composition according to (5), which can be injected through injection or spray.

(7) The adhesive hydrogel composition according to (6), which can be injected into living tissue through injection or spraying.

(8) The adhesive hydrogel composition according to (5), which has a function of suppressing an immune response.

After EGCG is oxidized, the hydrogel composition of the present invention can form covalent bonds with in vivo tissues in various ways, and thus has strong tissue adhesion ability.

In addition, the hydrogel composition of the present invention can achieve rapid cross-linking by using the strong oxidizing power of EGCG, and can be delivered in the form of injection through such rapid cross-linking, and can have strong tissue adhesion by the oxidation reaction of EGCG. Compared to fibrin glue and cyanoacrylate glue, which are often used in existing medical procedures, it shows superior ability in tissue adhesion, burst pressure, and liver hemostasis comparison experiments. In addition, the hydrogel composition of the present invention shows excellent tissue adhesion ability even in a wet environment.

In addition, the present invention can maximize tissue adhesion ability by introducing polyphenols that can maximize the effect of tyrosinase into the hydrogel and using polyphenols having four phenols in one unit.

In addition, the hydrogel composition of the present invention can react under physiological conditions, and since it is a biocompatible reaction that does not generate heat or by-products during the reaction, the success rate of hydrogel implantation can be increased.

In addition, the hydrogel composition of the present invention can be injected into the body in a minimally invasive way because the crosslinking reaction is completed within a few seconds and can be delivered in the form of injection through a syringe.

In addition, the hydrogel composition of the present invention has the ability to control the immune response to remove the radicals in the body through the radical removal ability of polyphenols and to induce an immune response to a minimum through this.

1 to 3 show that EGCG and tyrosine were introduced into hyaluronic acid, respectively (HA_E and HA_T, respectively), and then they were mixed in an appropriate ratio (HA_TE), and this was reacted with Streptomyces avermitilis-derived tyrosinase (SA_Ty). This is the result of confirming their crosslinking mechanism and confirming the appropriate ratio.
1 is a pictorial representation of the crosslinking mechanism of (HA_T) and (HA_TE) by (SA_Ty).
2A is a result of measuring the crosslinking time according to the ratio of (HA_E). 2B is a result of analyzing the degree of adhesion of the hydrogel to the mouse skin tissue according to the concentration of (HA_E).
FIG. 3A is an image of mouse heart and kidney tissue after cutting into two pieces and adhering using (HA_TE) hydrogel. 3B is a comparison image of the adhesive strength of a group in which (HA_TE) was reacted with (SA_Ty) and a group that did not react with (HA_TE).
Figure 4A is a diagram showing the adhesion ability test of HA_TE, fibrin glue and cyanoacrylate glue to mouse skin tissue. 4B is a graph showing the results of the adhesion test in terms of energy.
5A is a diagram illustrating a method for measuring burst pressure using a collagen tube. Figure 5B is a graph showing the burst pressure test results.
FIG. 6A is a hemostasis test diagram in which the amount of blood flowing out after a mouse liver tissue is wounded with a needle and then blocked with each material. 6B is a graph showing the results of a hemostasis test calculated by the amount of flowed blood.
7 is a result of comparative evaluation of the degree of immune response induction of HA_TE, fibrin glue, and cyanoacrylate glue in vitro and in vivo. Figure 7A is a diagram showing the mechanism by which EGCG suppresses the immune response. 7B is a result of measuring the amount of tissue necrosis factor (TNF) produced by immune cells after growing with each material. Figure 7C is a result of injecting each material into the subcutaneous tissue of the mouse, 3 days later, the serum of the mouse was extracted and the amount of immune-related cytokines present in the serum was measured.
Figure 8 is a comparison of tissue adhesion and skin regeneration ability using HA_TE, fibrin glue, and cyanoacrylate glue using mouse skin (native tissue) that was not treated with anything as a control by excising the skin of the mouse. It is the result. 8A and B are the results of histological analysis after 1 week and 2 weeks after the incised skin was adhered with each material. Figure 8A is the result of staining with hematoxylin and eosin (H&E, Hematoxylin and Eosin) to observe the morphology of the tissue, hematoxylin stains the cell nucleus, and eosin (Eosin) stains the cytoplasm. 8B is a result of observation of staining with Mason's trichrome (MTC). When the skin tissue is stained with Mason's trichrome, the collagen part is generally stained blue.

Hereinafter, the present invention will be described in more detail. However, the present invention may be changed and implemented in various forms, and is not limited to the embodiments described herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are those well known and commonly used in the art.

The present invention relates to a polymer in which EGCG (epigallocatechin gallate) is introduced into hyaluronic acid, alginate, chondroitin sulfate, chitosan or polyethylene glycol; and hyaluronic acid, alginate, chondroitin sulfate, chitosan or a polymer introduced with a phenol derivative in polyethylene glycol, and cross-linking using a tyrosinase derived from Streptomyces avermitilis. It provides a method for producing an adhesive hydrogel composition comprising, an adhesive hydrogel composition prepared thereby.

In the present invention, an EGCG duplex was introduced into the hydrogel in order to impart the hydrogel's rapid cross-linking reaction and immune response modulating ability.

The tyrosinase used as a catalyst for crosslinking in the present invention is derived from the genus Streptomyces, specifically, from Streptomyces avermitilis.

In addition, as the polymer used for preparing the hydrogel composition of the present invention, hyaluronic acid, alginate, chondroitin sulfate, chitosan or polyethylene glycol (PEG) may be used. Preferably, hyaluronic acid may be used.

As the phenol derivative, tyramine, tyrosine, 4-hydroxyphenylacetic acid or 3-(4-hydroxyphenyl)propionic acid may be used, preferably tyramine or tyrosine.

Another embodiment of the present invention is an adhesive hydrogel composition injectable through injection or spray. In particular, after mixing the EGCG-introduced polymer and the phenol derivative-introduced polymer with tyrosinase, it can be delivered into the living body by injecting or spraying it into a living tissue. In addition, the adhesive hydrogel composition has a function of suppressing an immune response, which can induce an immune response to a minimum, by removing radicals from the affected part of the body due to the radical removal ability of polyphenols.

In addition, the hydrogel composition of the present invention can be used as a medical material and can be applied to a biomaterial-based technology. For minimally invasive delivery of drugs and cells, the hydrogel composition of the present invention is a medical material and a functional biomaterial-based technology in that a phase change from a liquid to a solid phase should be made within a short time, and protection against external stimuli of the delivered material should be achieved. can be applied to In addition, the present invention can be applied to the development of drug delivery system technology and tissue engineering technology in that it can deliver drugs and hydrogels for tissue regeneration to damaged tissues or organs, and can minimize the induction of immune responses.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the contents of the present invention, and the present invention is not limited by the examples.

[Example]

Preparation Example 1: Preparation of Streptomyces avermitilis-derived tyrosinase (SA_Ty)

After the transformation by introducing the pET28a vector into which the Streptomyces avermitilis-derived tyrosinase (SA_ty) gene was inserted into the E. coli BL21(DE3) strain, the colonies obtained by applying to the LB solid medium were treated with 50 μg/mL kanamycin. (kanamycin) was inoculated into 3 mL of LB liquid medium and incubated for 8 hours in a shaking incubator at 37°C. For subculture, 50 μg/mL kanamycin and 500 μl of a seed culture medium were added to 50 mL of LB liquid medium and cultured in a shaking incubator at 37°C.

When OD ₆₀₀ (optical density at 600 nm) reached about 0.6, 0.02 mM IPTG or 0.2 mM IPTG, and 1 mM CuSO ₄ were added, and protein overexpression was induced at 37° C. for 20 hours. To obtain protein, cells were harvested by centrifugation at 4,000 rpm for 10 minutes, and 5 mL of 50 mM Tris-HCl buffer (pH 8.0) was added to wash the cells. After adding 5 mL of 50 mM Tris-HCl buffer (pH 8.0) again, cell disruption was performed using a sonicator (Vibra & cell, USA). Then, the cell lysate was dispensed into a 1.7 mL tube and centrifuged at 16,000 rpm for 30 minutes to obtain a cell extract containing protein.

This cell extract was purified by His-tag using a Ni-NTA column. First, Ni-NTA activity was induced using 1 column volume of pre-binding buffer (5 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl buffer), and the cell extract was applied to a Ni-NTA column. After binding the tyrosinase to the column, impurities not strongly bound to the Ni-NTA column using twice the column volume of wash buffer (25 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl buffer) were removed. Finally, tyrosinase was separated from the Ni-NTA column using elution buffer (250 mM imidazole, 50 mM Tris-HCl), and then 50 mM Tris using a 10k filter to dilute the imidazole to 1/2500 level. The buffer was replaced with -HCl to obtain purified tyrosinase. The purified tyrosinase was used in the cross-linking reaction shown in Preparation Examples and Examples below.

Preparation Example 2: EGCG duplex production

EGCG was dissolved in a mixed solution of THF (tetrahydrofuran, Sigma-Aldrich) and MSA (methanesulfonic acid, Sigma-Aldrich). Then, a mixed solution of DA (2,2-diethoxyethylamine, Sigma-Aldrich), THF, and MSA was added and reacted at room temperature for 24 hours. Thereafter, all solvents were removed using a rotary evaporator and dried at low pressure for 24 hours. Then, it was dissolved in distilled water, and the EGCG duplex was extracted using ethyl acetate.

Preparation Example 3: Synthesis of HA_T and HA_E

Hyaluronic acid (Lifecore Biomedical, LLC, 40-64 kDa) was dissolved in primary distilled water at 1% (w/v). The above solution by setting the molar concentration ratio of EDC (ethyl(dimethylaminopropyl) carbodiimide, ThermoFisher): Sulfo-NHS (sulfo-N-hydroxysulfosuccinimide, ThermoFisher): tyramine hydrochloride (Sigma-Aldrich) to 1:1:1 , and reacted at room temperature for 24 hours. After the reaction, dialysis was performed for two days using a dialysis membrane (Snakeskin, Cutoff MW: 3kDa, ThermoFisher), and the remaining solution was freeze-dried to obtain HA_T.

Hyaluronic acid (Lifecore Biomedical, LLC, 40-64 kDa) was dissolved in 1% (w/v) in a solution in which primary distilled water and dimethylformamide were mixed in a 10:1 volume. Then, the molar concentration ratio of EDC: Sulfo-NHS: EGCG was set to 1:1:2, dissolved in the above solution, and reacted at room temperature for 24 hours. Then, using hydrochloric acid and brine, the synthesized hyaluronic acid-EGCG conjugate (HA_E) was purified. After the purified HA_E was dialyzed using a dialysis membrane for one day, the remaining solution was freeze-dried to obtain HA_E.

Preparation Example 4: HA_TE hydrogel production

A crosslinking reaction was induced by adding 200 nM of SA_Ty to a solution in which a 4% HA_T solution and a 2% HA_E solution were mixed at a volume ratio of 1:1.

Example 1. Establishment of the composition of HA_T and HA_E

In order to introduce EGCG into HA, an EGCG duplex was synthesized and introduced into HA. EGCG is composed of 4 phenolic structures, and the EGCG duplex is composed of 8 phenolic structures, so the reactivity between HA_E is very low due to steric hindrance. EGCG in HA_E is oxidized very rapidly, but there is no reaction between HA_E. For this reason, HA_T, which can avoid steric hindrance, is combined in a certain ratio to maximize the reactivity by SA_Ty. The final concentration of HA_T was fixed at 2%, and the concentration of HA_E was increased from 0.1 to 1% to measure the crosslinking time through a vial tilting test.

The vial tilting test was performed as follows: 1 ml of each HA_T 2%, HA_TE 0.1%, HA_TE 0.5%, HA_TE 1.0% solution was put into a 10ml glass vial, and a vial once every 5 seconds. After tilting the vial, the time was measured when there was no flow of solution in the vial.

As a result, it can be seen that the reaction rate increases sharply as the concentration of HA_E increases (see FIG. 2A and Table 1 below).

Meanwhile, in the same manner as above, the final concentration of HA_T was fixed at 2%, and the concentration of HA_E was increased from 0.1 to 1% to measure the degree of adhesion energy of the hydrogel to mouse skin tissue according to the concentration of HA_E as follows. :

1. The mouse skin was cut into 1 cm x 3 cm wide.

2. 50 ul of hydrogel was applied between two mouse skins to fill a space of 1 cm x 1 cm.

3. After 5 minutes of gelation, a stress-strain curve was obtained while pulling at a speed of 1 mm/min through a universal tensile tester.

4. The adhesion energy was calculated based on the data obtained through the stress-strain curve.

As a result, it can be seen that the degree of adhesion energy is higher in HA_TE than in HA_T, and as the concentration of HA_E increases, the adhesion work with mouse skin tissue increases (see FIG. 2B and Table 2 below). . In Table 2 below, σmax is the maximum pressure shown when the hydrogel does not withstand the pressure and breaks during the compression test of the hydrogel, and εmax is the hydrogel that does not withstand the pressure and cracks during the compression test. is the change value of the strain shown when E is the Young's modulus of the hydrogel, and the failure force is the maximum adhesive force shown when the adhesion is broken during the adhesion test of the hydrogel.

This also shows that the HA_TE hydrogel can react within a few seconds and strongly interact with the mouse skin tissue to achieve strong adhesion.

Example 2: Evaluation of adhesion of HA_TE after dissection of actual organs

After cutting mouse heart and kidney tissue into two pieces, the two pieces were adhered using HA_TE hydrogel.

As a result, it was confirmed that when attached with HA_TE, it was stably adhered (see FIG. 3A).

Example 3: Adhesion evaluation when reacted with SA_Ty

In vitro, the adhesive strength of the group in which (HA_TE) was reacted with (SA_Ty) and the group that did not react with (HA_TE) were compared and evaluated. Specifically, adhesion was evaluated as follows:

1. A 2 cm x 2 cm size mouse skin was fixed on a slide glass with an instant adhesive.

2. Divided into a group that was reacted with (SA_Ty) and a group that did not react with (HA_TE), each was applied to the skin of the mouse, and two slide glasses were attached.

3. After 5 minutes of reaction, weights of 10, 50, and 100 g were lifted to check whether the adhesive force was maintained.

As a result, it was confirmed that the group in which (HA_TE) was reacted with (SA_Ty) could withstand a weight of up to 100 g, and in the group that did not react with (SA_Ty), it was confirmed that the adhesive strength was not maintained even at a weight of 10 g. (see Fig. 3B).

Example 4: Comparative evaluation of adhesion between HA_TE and a representative adhesive

Adhesive ability with fibrin glue, cyanoacrylate glue and HA_TE was compared and evaluated.

1) Adhesive ability test to mouse skin tissue

The mouse skin tissue was adhered using fibrin glue, cyanoacrylate glue and HA_TE, respectively, and the degree of adhesion energy was measured (see Fig. 4A). The test proceeded as follows:

1. The mouse skin was cut into 1 cm x 3 cm wide.

2. 50 ul of each material was applied between two mouse skins to fill a space of 1 cm x 1 cm.

3. After 5 minutes of gelation, the sample was immersed in saline (PBS, Phosphate-buffered Saline) for 5 minutes.

4. While pulling at a speed of 1 mm/min through a universal tensile tester, a stress-strain curve was obtained.

5 Adhesion energy was calculated based on the data from the stress-strain curve.

As a result, in the adhesion test with mouse skin, cyanoacrylate glue showed the highest level. However, it was confirmed that the adhesion of the cyanoacrylate glue was markedly reduced when exposed to a wet environment through the step of immersing the sample in saline for 5 minutes as described above. HA_TE showed a higher degree of adhesion than fibrin glue, and it was confirmed that adhesion was maintained even in a wet environment (see FIG. 4B and Table 3 below).

In Table 3 below, σmax is the maximum pressure shown when the hydrogel does not withstand the pressure and breaks during the compression test of the hydrogel, and εmax is the hydrogel that does not withstand the pressure and cracks during the compression test. is the change value of strain shown when, E is the Young's modulus of the hydrogel, and the failure force is the maximum adhesive force shown when the adhesion is broken during the adhesion test of the hydrogel.

2) burst pressure test

The burst pressure test is a test that can determine whether or not it is applied to tissues to which strong pressure is transmitted, for example, blood vessels, heart, liver, and the like. In order to withstand the burst pressure, fast crosslinking, strong adhesion, and the elasticity of the material itself are important.

The test proceeded as follows (see Figure 5A):

1. A pressure device was fixed at the bottom of the collagen tube. After that, after puncturing the tube, each material was applied.

2. After 5 minutes of gelation, water was poured into the tube to measure the burst pressure.

As a result, HA_TE showed a superior ability to withstand burst pressure compared to other materials (see Fig. 5B).

3) liver hemostasis test

The liver hemostasis test is a test that can determine whether the tissue can be sutured even when blood flows after making a wound on live mouse liver tissue with a syringe.

The test proceeded as follows (see Figure 6A):

1. After anesthetizing the mouse, an abdominal incision was made to expose the liver.

2. Hepatic hemorrhage was induced by using an 18G needle after fixing the filter paper on the lower part of the liver.

3. After suturing the bleeding site with each material, the weight of the filter paper was measured to measure the amount of blood absorbed.

4. The amount of bleeding was calculated by calculating the difference between the weight of the original filter paper and the weight of the filter paper in which blood was absorbed.

When the amount of flowed blood was measured after suturing with each material, it was observed that the amount of blood was the lowest in HA_TE (see FIG. 6B).

Example 5: Comparative evaluation of immune induction ability between HA_TE and a representative adhesive

To compare the immune-inducing ability of HA_TE and representative adhesives (fibrin glue, cyanoacrylate glue), immune cells, RAW 264.7 cells, were grown together with each material, and tissue necrosis factor released when the cells induce an immune response. The amount of (TNF) was measured. In addition, as an additional comparison group, a group treated with lipopolysaccharide (LPS) that stimulates RAW 264.7 cells was used. The cyanoacrylate glue excreted a significantly higher level of TNF than the LPS-treated group, and the fibrin glue showed a slightly lower level than the LPS-treated group. However, HA_TE showed similar values to the group not treated with LPS (see FIG. 7B). This shows that HA_TE can minimally stimulate immune cells and suppress immune stress.

In addition, each material was injected into the mouse subcutaneous tissue and serum was separated 3 days later to analyze the amount of immune-related cytokines in the serum. The test proceeded as follows:

1. 100 ul of each material was injected into the subcutaneous fat of the mouse.

2. Three days after transplantation, blood was collected from the sinus behind the eye.

3. After separating serum from blood, IL-6, IL-10, MCP-1, IFN-γ, TNF, and IL- using BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Bioscience, USA) The amount of 12p70 was analyzed by FACS.

As a result, the amount of most immune-related factors was the lowest in HA_TE, which is similar to the group injected with saline (PBS, Phosphate-buffered Saline) (see FIG. 7C). It can be seen that HA_TE hydrogel induces an immune response in the body to a minimum and suppresses the immune response by removing radicals from the wounded area.

Example 6: Evaluation of adhesion ability and tissue regeneration ability of each material in mouse skin incision model

The mouse skin incision model was histologically analyzed after 1 week and 2 weeks. The test proceeded as follows:

1. After anesthetizing the mouse, a 1 cm incision was made in the dorsal subcutaneous tissue.

2. The incision was sutured using each material. As a control group, untreated mouse skin (native tissue) was used, a negative control group was an untreated group, and a positive control group was a group sutured (suture).

3. After 1 or 2 weeks, the tissue of the sutured part was collected and fixed in paraformaldehyde (sigma-aldrich, St.Louis, MO) for 24 hours.

4. The fixed samples were washed with saline (PBS, Phosphate-buffered Saline) for 5 minutes, and then dehydrated with 50, 75, 90, 95, and 100% ethanol solutions, respectively.

5. The dehydrated samples were embedded in paraffin solution.

6. The tissue embedded in paraffin was cut to a thickness of 10 μM using a multipurpose slicer.

7. The cut samples were observed by staining with H&E (hematoxylin and eosin, sigma) or with MTC (Masson's trichrome).

For reference, hematoxylin stains the cell nucleus, and eosin stains the cytoplasm. In addition, in MTC (Masson's trichrome) staining, when the skin tissue is generally stained, the collagen part is stained blue.

As a result of histological analysis after 2 weeks of the mouse skin incision model, the HA_TE group showed the most similar shape to the actual skin tissue. Specifically, when HA_TE was used, the skin tissue was regenerated well, and pores and sweat glands existing in the skin tissue were completely regenerated.

On the other hand, the group using the suture showed the next best regeneration, and although all skin tissues were closed, pores and sweat glands were still regenerated.

In addition, fibrin glue and cyanoacrylate glue were less finished tissue remodeling, and strong fiber formation symptoms were observed in the skin tissue (see FIG. 8 ). This shows that collagen is not suitable for skin adhesive as a symptom of replacing actual skin in the incised area rather than complete skin regeneration.

Claims (8)

polymers introduced with EGCG (epigallocatechin gallate) in hyaluronic acid, alginate, chondroitin sulfate, chitosan or polyethylene glycol; and
Hyaluronic acid, alginate, chondroitin sulfate, chitosan or polyethylene glycol is blended with a phenol derivative or a polymer introduced with phenol, which is tyramine, tyrosine, 4-hydroxyphenylacetic acid or 3-(4-hydroxyphenyl)propionic acid,
Streptomyces avermitilis ( Streptomyces avermitilis ) A method for producing an adhesive hydrogel composition comprising the step of cross-linking using a tyrosinase derived. The method of claim 1,
A method for producing an adhesive hydrogel composition comprising a polymer in which EGCG is introduced into hyaluronic acid and a polymer in which a phenol derivative is introduced into hyaluronic acid. delete The method of claim 1,
The phenol derivative is tyramine or tyrosine, the method for producing an adhesive hydrogel composition. [Claim 5] The adhesive hydrogel composition prepared by the method of any one of claims 1, 2 and 4. 6. The method of claim 5,
Injectable through injection or spray, the adhesive hydrogel composition. 7. The method of claim 6,
An adhesive hydrogel composition that can be injected into living tissue through injection or spraying. 6. The method of claim 5,
An adhesive hydrogel composition having a function of suppressing an immune response.

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